REGULATION OF TRANSDUCIN GTPASE ACTIVITY IN BOVINE ROD OUTER SEGMENTS

The photoreceptor G-protein, transducin, belongs to the class of heter otrimeric GTP-binding proteins that transfer information from activate d seven-span membrane receptors to effector enzymes or ion channels. L ike other G-proteins, transducin acts as a molecular clock. It is acti vated by photoexcited rhodopsin which catalyzes the exchange of transd ucin-bound GDP for GTP and then stays active until bound GTP is hydrol yzed by an intrinsic GTPase activity. Our previous study on the compon ents of the amphibian phototransduction cascade (Arshavsky, V.Y., and Bownds, M. D. (1992) Nature 357, 416-417) has shown that transducin GT Pase can be significantly accelerated by the target enzyme, cGMP phosp hodiesterase (PDE), and more specifically its gamma-subunit (PDE(gamma )). Here we report that an analogous mechanism is present in bovine ph otoreceptors. Addition of recombinant PDE(gamma) to the test photorece ptor membranes which retain transducin but are depleted of endogenous PDE causes a significant acceleration of transducin GTPase activity. A similar effect was observed with the PDE holoenzyme, but not with the complex of PDE alpha and beta-subunits prepared by a limited proteoly sis of PDE with trypsin. The activating effect of PDE(gamma) is increa sed as test membrane concentration increases, exceeding 20-fold at rho dopsin concentrations over 80 mu M and approaching the rate of the pho tore sponse turnoff. This suggests either that photoreceptor membranes contain a further factor which is essential for PDE-dependent regulat ion of transducin-bound GTP hydrolysis or that components of the photo transduction cascade interact in a cooperative manner. We also report that the GTPase-activating epitope is located within the C-terminal th ird of PDE(gamma): the peptide corresponding to the 25 C-terminal amin o acid residues of PDE(gamma) can accelerate transducin GTPase almost as well as the full length PDE(gamma). A part of the GTPase activating epitope is located within the 3 C-terminal amino acid residues: the t runcation PDE(gamma) mutant lacking these residues accelerates transdu cin GTPase considerably less than the whole length PDE(gamma).